Project Summary/Abstract There are no effective countermeasures for the acute Radiation GI Syndrome (RGS). Studies proposed here will provide support for our ongoing effort to develop an anti-ceramide Ab as a mechanism-based approach to mitigate RGS lethality. The RGS results from destruction of crypt/villus units, loss of mucosal integrity, and infection by resident enterobacterial flora. Recent data indicate RGS pathophysiology involves depletion of a pool of small intestinal stem cells (ISCs) residing at the bottom of the Crypts of Lieberkhn termed crypt base columnar cells (CBCs). We recently showed CBCs to be more radioresistant than transit amplifying progenitors or differentiated villus cells, protecting themselves effectively by homologous recombination. Further, CBC depletion is biphasic post-ionizing radiation (IR) as apoptosis occurs in the first 24h during growth arrest, followed by mitotic death at 24-48h during the rapid regenerative phase that ensues once CBCs begin re- cycling. CBC depletion by apoptosis and mitotic death precede physical dissolution of crypts between Day 2.5- 3.0 post IR. Further, CBC survival at Day 2 predicts both crypt regeneration at Day 3.5 measured by the Clonogenic Assay of Withers and Elkind, and RGS lethality. In addition to direct ISC damage, our lab proposed that IR-induced injury to small intestinal microvasculature plays a prominent role in outcome. Specifically, we propose IR causes release within min of acid sphingomyelinase (ASMase) to the outer endothelial plasma membrane, where it finds its substrate sphingomyelin and generates the pro-apoptotic second messenger ceramide. Ceramide assembles a signaling platform on the endothelial surface that mediates apoptosis, and the coupling of microvessel injury to direct ISC damage coordinately determines ISC survival. Further, we recently reported our anti-ceramide 2A2 Ab binds ceramide on the irradiated endothelial surface, preventing formation of ceramide-rich platforms required for endothelial death, thereby protecting mice against RGS lethality. In unpublished studies, we show a 2nd prolonged wave of endothelial apoptosis occurs post high dose IR accompanied by ASMase secretion into the systemic circulation of mice that persists for 4 days indicative of ongoing vascular damage/dysfunction. Here, in 3 specific aims we will detail this vascular syndrome, determine its role in CBC demise post IR, and show that a single chain variable fragment (scFv) of anti-ceramide Ab injected s.c. attenuates it, mitigating CBC depletion and improving mouse survival. Using new genetic and biologic tools we will formally show that the scFv mitigates the RGS by inhibiting ceramide generated on endothelial but not tumor parenchymal cells. Once the mechanism of scFv mitigation is delineated we will combine it with IL-22, an agent recently shown by us to improve CBC regeneration after IR. There is currently a dearth of information regarding the immediate post-radiation milieu of the small intestinal mucosa. Studies here should fill in gaps regarding impact of microvascular damage on evolving mucosal injury and increase our knowledge to optimize anti-ceramide strategies to mitigate RGS lethality.